The PX domain: a new phosphoinositide-binding module (original) (raw)
Related papers
PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox
Nature Cell Biology, 2001
The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b 558 (gp91 phox and p22 phox ) and three soluble factors (GTP-Rac, p47 phox and p67 phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40 phox has subsequently been discovered as a binding partner for p67 phox (ref.
The NOXO1β PX Domain Preferentially Targets PtdIns(4,5)P2 and PtdIns(3,4,5)P3
Journal of Molecular Biology, 2012
NOXO1β [NOXO1 (Nox organizer 1) β] is a cytosolic protein that, in conjunction with NOXA1 (Nox activator 1), regulates generation of reactive oxygen species by the NADPH oxidase 1 (Nox1) enzyme complex. NOXO1β is targeted to membranes through an N-terminal PX (phox homology) domain. We have used NMR spectroscopy to solve the structure of the NOXO1β PX domain and surface plasmon resonance (SPR) to assess phospholipid specificity. The solution structure of the NOXO1β PX domain shows greatest similarity to that of the phosphatidylinositol 3-kinase-C2α PX domain with regard to the positions and types of residues that are predicted to interact with phosphatidylinositol phosphate (PtdInsP) head groups. SPR experiments identify PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 as preferred targets of NOXO1β PX. These findings contrast with previous lipid overlay experiments showing strongest binding to monophosphorylated PtdInsP and phosphatidylserine. Our data suggest that localized membrane accumulation of PtdIns(4,5)P 2 or PtdIns(3,4,5)P 2 may serve to recruit NOXO1β and activate Nox1.
Journal of Biological Chemistry, 2009
Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new ␣2helix and a much longer ␣3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.
Proceedings of the National Academy of Sciences, 2003
Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 phox , because protein kinase C treatment of the wild-type p47 phox but not of a mutant protein with the S303͞ 304͞328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 phox nor lipid-bindingdefective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.
Modular phosphoinositide-binding domains – their role in signalling and membrane trafficking
Current Biology, 2001
The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.
Increased mobility in the membrane targeting PX domain induced by phosphatidylinositol 3-phosphate
Protein Science, 2006
Phosphoinositides (PIs) arec oncentrated in specific subcellular membranes in order to recruit and regulate cytosolic proteins responsible for vesicular trafficking, cytoskeletalr earrangement, and eukaryotic cell growth,d ifferentiation, ands urvival.P hoxh omology( PX) domainsa re foundi n proteins that are integral players in endocytic pathways. Fore xample, Va m7p is targeted by itsP X domain to phosphatidylinositol 3-phosphate[ PtdIns(3)P] in the yeast vacuole, where it interacts with other SNAREproteinsand GTPasesofthe vesicular membrane fusion machinery.Although several PX structures have been solved, therole of dynamics in their interactions with membrane lipids is unclear. Here, we present the firstd etailed characterizationo ft he backbone dynamics of aP Xd omain,t hato f Va m7p, in thep resence anda bsence of itsl igand.T he structure appears to tumble more rapidly in solution upon binding PtdIns(3)P,r evealingaconformational change that includes adjustments in the flexiblem embrane insertion loop (MIL). The flexibilities of theM IL andd omain termini are pronounced in both states,w hile the a 1a nd a 2h elicesa re rigid. Dynamic effects ares preada cross the binding pocket, withP tdIns(3)P inducing altered mobility of different residues on multiple timescales, including ashift in the MIL to slower timescale motions. Theb oundstate is more dynamic overall, particularlyinthe b -sheet lobe, which packs against the ligand's 3-phosphate. Thus, theinduced dynamic andstructuraleffects are transducedfromthe buried heart of the binding pocket in thehelical lobe throught he b -sheet lobe to thee xposed surface of theb ilayer-inserted protein.
The EMBO Journal, 2002
p47 phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47 phox PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different speci®cities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40 phox , while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipidbinding pockets radically increases membrane af®nity. Strikingly, measurements for full-length p47 phox show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-speci®c mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47 phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater af®nity than the isolated PX domain.
Journal of cell science, 2014
Sorting nexin 27 (SNX27) controls the endosomal to cell-surface recycling of diverse transmembrane protein cargos. Critical to this function is the recruitment of SNX27 to endosomes through the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by the phox-homology (PX) domain. In T cells, SNX27 is polarized to the immunological synapse (IS) in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide lipid-binding capabilities of full-length SNX27, and discovered a novel PtdInsP binding site within the C-terminal 4.1/ezrin/radixin/moesin (FERM) domain. This binding site showed a clear preference for di and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the IS of activated T-cells cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FER...
PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation
The EMBO Journal, 2006
The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX domains of two oxidase components, p47(phox) and p40(phox), are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX domain of their p40(phox) gene, which selectively prevents binding to PtdIns3P. p40(phoxR58A/R58A) embryos do not develop normally but p40(phoxR58A/-) mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40(phox+/-) siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P-binding to p40(phox). p40(phoxR58A/-) mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.